Deciphering the molecular heterogeneity of spiral ganglion neurons by single-cell gene expression profiling.

The human ear is an extraordinary sensory organ, in which sensory cells and their nerve connections are able to analyse an impressive range of sound frequencies and intensities. The role of the sensory hair cells is to convert sound information from the outside world into electrical signals that are sent to the brain via specialized nerve fibres, allowing us hear speech and music. The development of the auditory organ, the cochlea, is an extremely ordered process, which allows to build sensory cells and nerve connections that, for example, respond preferencially to either low- or high-frequency sound depending on their location along the sensory organ.

Age-related hearing impairment (ARHI) is a complex disorder caused by a combination of genetic and environmental factors. Noise exposure is the major environmental factor that causes ARHI. It is clinically very difficult to distinguish between these two most common forms of hearing impairment: noise-induced and age-related hearing impairments (NIHI and ARHI respectively). The large impact of NIHI and ARHI on human health is caused by the continuous increase in the average lifespan of the population, and by the fact that our ears are not well adapted to cope with long-lasting exposure of loud sounds characteristic of modern society. Currently, the only option available to ameliorate hearing loss is using hearing aids and cochlear implants, which are beneficial but they are far from restoring normal hearing. The problem is that we still know very little about the biological mechanisms causing NIHI and ARHI to be able to develop effective alternative treatments to either prevent or cure this disease.

Until very recently the sensory cells have been considered the most vulnerable elements to aging and noise exposure but recent finding have shown that their nerve connections are more easily damaged during insults. In the adult auditory system, each sensory cell in the ear (inner hair cell) is contacted by multiple nerve connections that are anatomically and physiologically diverse, and as such able to carry a different sound intensity and frequencies to the brain. In particular, it has been suggested that the nerve connections having the highest detectable sound intensities seem more vulnerable to noise and aging, resulting in their specific damage. However, there is no direct evidence as to why these specific nerve connections are predominantly affected by aging and/or noise exposure as compared to those responding to lowest detectable intensity sound. Therefore, the ability to identifying genetic factors and molecules that render these nerve connections more susceptible to aging and/or noise trauma is essential for devising early diagnostic, intervention and/or treatments for both ARHI & NIHI. Identifying the genetic factors and molecules in humans has been hampered by many inherent difficulties: 1) not all individuals with the same genetic defects have the same clinical presentations, probably depending on the intensity and the duration of the noise exposed to; 2) similar environmental exposures sometimes have different effects on individuals, probably because of differences in their underlying genetic makeup.

For these reasons we will address this important aspect of human biology by studying gene-noise interaction in mice where both factors can be controlled and we know that the structure and physiology of the ear is similar to that of humans. In the proposed project, we will combine expertise in genetic and physiology to evaluate gene expression and function in the nerve connections. Our approach will generate new mouse models to address why a specific population of nerve connections is selectively damaged to noise and aging. These steps are important towards understanding the etiology of human noise-induced and age related hearing impairment (long-term goal), and will take us closer to the goal of developing a suitable therapeutic intervention to treat patients.

In both noise and age related hearing impairment synapses degeneration precedes hair cell loss and threshold shift. Understanding why subtypes of synapses and their corresponding type I spiral ganglion neurons (SGNI) die requires the knowledge of genes that are expressed in individual neurons and that contribute to their differential vulnerability to noise. Our main goal in this study is to determine identity-specific molecular profiles and define genes that distinguish SGNI subtypes.

My laboratory is now in a unique position to make these discoveries. We have recently generated and used a transgenic mouse with the unique feature of fluorescently labeled SGNIs to sort single neurons. Single cell gene-expression profiling was used to distinguish and to characterize different SGNI subtypes. Our pilot studies demonstrate feasibility of our approach. We have identified specific, potential marker genes that specifically label distinct subpopulations of presumptive low and high threshold neurons. We will select validated and highly differentially expressed genes to generate subtype-specific reporter mice that will also allow us to conditionally eliminate presumptive low and high threshold SGNI. These mouse models will be utilized to address physiologically important questions such as 1) what are the molecular and cell biological differences in physiologically distinct SGNI subgroups? & 2) what are the mechanisms underlying differential vulnerability of low versus high threshold SGNI to noise trauma?
Using cutting edge technologies and the opportunity to work with world experts in the field at the Sheffield University & MRC Harwell, I am confident that we will contribute to the identification of genetic factors involved in the cochlear response to aging and/or noise trauma. Knowledge of these mechanisms is essential for devising early diagnostic, intervention and/or treatments for auditory synaptopathy, and consequently for age-related and noise- induced hearing loss.

Academic Impact:
The proposed work will provide an in depth understanding of the molecular and cellular mechanisms underlying the different auditory neurons innervating the mammalian cochlea and their vulnerability to noise and, ultimately, to aging. Therefore, this project will be of great interest not only to the auditory scientists interested in cochlear function and different forms of hearing loss, but also to a large proportion of neuroscientists interested in neuronal network function and coding of sensory signals.
We will continue to disseminate our results in peer-reviewed, general science publications and conference presentations. We also expect that the results produced will result in invited talks and seminar at leading international institutions, which will be given by the PI and PDRAs. We will also present the results to leading international conferences focused on neuroscience and physiology. In addition we are proposing to organize an international symposium at the University of Sheffield that will bring together PIs interested in auditory signal processing. The principle applicants will present results to meetings focused on neuroscience, physiology and hearing research, which will ensure that we reach a wide scientific audience.

Societal and economic Impact:
People affected by hearing loss struggle to communicate with the public and even their own families, which ultimately lead to their social isolation and cognitive impairment. Noise and aging related hearing loss is the most common sensory disorder in humans, which is mainly dictated by the continuous increase in the average lifespan of the population, and also to the fact that our ears are ill-adapted to cope with long-lasting exposure of loud sounds characteristic of modern society.
The proposed project will directly provide new insights into the cause of these forms of hearing loss, which will lead to the identification of new target molecule for the future development of therapies for the disease. We will also inform the general public via several routes: every year the University of Sheffield organizes together with Action on Hearing Loss an open meeting attended by elderly people to discuss in lay terms our scientific work on age- and noise-induced hearing loss. We also present our findings in lay terms to the public using the several activities organized at the University of Sheffield, such as Discovery night and Science Week.

Post-doctoral scientist:
The proposed work combines a wide-range of physiological and molecular biological techniques using in vitro and in vivo models. Combined with the impact on clinical and social health, this will provide an excellent training for young PDRAs.
Postdoc will receive an expensive training not only via the wide range of expertise present in the hearing group at Sheffield, but also via the mouse-genetic training capabilities at the MRC Harwell. The hearing research group at Sheffield work very closely to that at MRC Harwell with joint projects and regular lab meetings.

Undergraduates at the University of Sheffield:
This research will also have a great impact on undergraduates, with the aim to recruit the next generation of scientists. The number of active hearing research PIs in the UK has been steadily decreasing over the last few years, resulting in the UK falling behind compare to other European countries.
The University of Sheffield runs a 3rd year module in Sensory Neuroscience, which normally attracts top class students. This module is fully research-driven, and as such we share the latest findings from our projects to the students with the additional aim to attract some of them into the hearing field. Since it has been implemented 2 years ago, we already attracted two top-class students in the field.